Induction of Rhesus Keratinocytes into Functional Ameloblasts by Mouse Embryonic Dental Mesenchyme

Fast progresses in stem cell-based tooth tissue engineering have been achieved in recent years in several animal models including the mouse, rat, dog, and pig. Moreover, various postnatal mesenchymal stem cells of dental origin have been isolated and shown capable of differentiating into odontoblasts and generating dentin. Meanwhile, human keratinocyte stem/progenitor cells, gingival epithelial cells, and even iPSC-derived epithelium have been demonstrated to be able to differentiate into functional ameloblasts. Translational medicine studies in the nonhuman primate are irreplaceable steps towards clinical application of stem cell-based tissue engineering therapy. In the present study, we first examined the epithelial stem cell markers in the rhesus skin using immunostaining. Keratinocyte stem cells were then isolated from rhesus epidermis, cultured in vitro, and characterized by epithelial stem cell markers. Epithelial sheets of these cultured keratinocytes, which were recombined with E13.5 mouse dental mesenchyme that possesses odontogenic potential in the presence of exogenous FGF8, were induced to differentiate into enamel-secreting ameloblasts. Our results demonstrate that in the presence of appropriate odontogenic signals, rhesus keratinocytes can be induced to gain odontogenic competence and are capable of participating in odontogenesis, indicating that rhesus keratinocytes are an ideal epithelial cell source for further translational medicine study of tooth tissue engineering in nonhuman primates.

Dissociated mouse tooth germ epithelial cells retain the expression of tooth developmental genes during reaggregation process / 生物工程学报

Generation of bio-engineered teeth by using stem cells will be a major approach for bioengineered implantation. Previous studies have demonstrated that dissociated tooth germ cells are capable of generating a tooth after reaggregation in vitro. However, the cellular and molecular mechanisms underlying this tooth regeneration are not clear. In this study, we dispersed E13.5 molar germ into single cells, immediately reaggregated them into cell pellet, then grafted the reaggregates under mouse kidney capsule for various times of culture. We investigated the morphogenesis and the expression of several developmental genes in dental epithelial cells in reaggregates of tooth germ cells. We found that dissociated tooth germ cells, after reaggregation, recapitulated normal tooth developmental process. In addition, dissociated dental epithelial cells retained the expression of Fgf8, Noggin, and Shh during reaggregation and tooth regeneration processes. Our results demonstrated that, despite of under dissociated status, dental epithelial cells maintained their odontogenic fate after re-aggregation with dental mesenchymal cells. These results provided important information for future in vitro generation of bio-engineered teeth from stem cells.

Construction of rat CXCR4 gene lentiviral RNA interference vector and its expression in mesenchymal stem cells / 生物工程学报

To construct the lentiviral RNA interference (RNAi) vector of rat CXCR4 gene, three target sequences were selected according to rat CXCR4 mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After phosphorylation and annealing, these double-strand DNA were cloned to Bgl II and Hind III sites of pSUPER. Then the product pRiCXCR4 was confirmed by electrophoresis and sequencing. Next, CXCR4 shRNA was cloned to a transfer vector of lentivirus, pNL-EGFP, and pNL-RiCXCR4-EGFP was produced. It was confirmed by digestion and sequencing that CXCR4 shRNA expression structure was correctly cloned to pSUPER and pNL-EGFP respectively. Three plasmids, pNL-RiCXCR4-EGFP, pHELPER and pVSVG were cotransfected into 293T to package lentivirus particles. The functional titer of obtained virus was determined by flow cytometry after transduction in 293T, the resulting functional titer of unconcentrated virus and concentrated virus were 6.4 x 10(4) TU/mL and 6.9 x 10(6) TU/mL respectively. After the rat mesenchymal stem cells (rMSCs) were transduced with the constructed lentiviral vectors, real-time RT-PCR, Western blotting and flow cytometry were used to evaluate the level of CXCR4 expression. Compared with control group, the CXCR4 mRNA expression were obviously suppressed in all three experimental groups (rMSCs-CXCR4a, rMSCs-CXCR4b, rMSCs-CXCR4c), especially the expression rate in rMSCs-CXCR4b group was reduced by 95.6%. The RNAi lentivirus vector of rat CXCR4 gene has been constructed successfully. This greatly facilitates the further studies such as evaluation the role of CXCR4 in rMSCs recruitment to damaged tissue.

Therapeutic efficacy of lentiviral vector mediated BDNF gene-modified MSCs in cerebral infarction / 生物工程学报

Pretreatment with brain-derived neurotrophic factor (BDNF) reduces ischemic damage after focal cerebral ischemia, and bone marrow mesenchymal stem cells(MSCs) were reported to ameliorate functional deficits after stroke in rats. Here we investigate the synergistically therapeutic effects of BDNF gene-modified MSCs on cerebral infarction. We transfected MSCs with the BDNF gene using a lentivirus-based system and investigated whether the BDNF-modified MSCs contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. Compared to untreated rats, rats that received both MSCs and BDNF-MSCs showed significantly more functional recovery. The difference in modified neurological severity score(mNSS) was statistically significant (P < 0.001). Recovery was better in BDNF-MSCs than in MSCs (P < 0.001). At the second week and second month after the systemic delivery of blank vector-modified MSCs and BDNF-modified MSCs, the treated rats exhibited more significant recovery than the control, including the accumulation and living of enhanced green fluorescence protein (EGFP)-positive cells in the infarct area and surrounding areas, neuron-like changes, expression of surface markers of neural cells, and a large amount of BDNF expression in the BDNF-MSCs-treated group. Our findings suggest that BDNF-gene-modified rMSCs can migrate to surrounding areas of the cerebral infarction lesion, differentiate into neural cells, and survive for extended periods. With the synergy of BDNF, they may promote the recovery of the neurological function following cerebral infarction and represent a new strategy for stem cell-based therapy.

Construction of lentiviral vector carrying the Ang-1 gene and its expression in the rMSCs / 中国病理生理杂志

AIM: To construct lentiviral vector carrying the angiopoietin-1(Ang-1) gene,and make it express Ang-1 in the rat mesenchymal stem cells(rMSCs).METHODS: The cDNA encoding the CDS of Ang-1 gene was obtained from the placenta of the adult Fisher 344 rats with RT-PCR.After digestion with restrication endonuclease,the Ang-1 gene was recombined to construct the transfer plasmid PNL-Ang1-IRES2-EGFP.The three-plasmid system of lentiviral vector was consisted of PNL-Ang1-IRES2-EGFP,the packaging plasmid HELPER,and the envelope plasmid VSVG,which were co-transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles.The rMSCs were infected by obtained lentiviral particles.The insertion of Ang-1 gene was detected by PCR,the mRNA expression of Ang-1 in rMSCs was detected with RT-PCR,the protein expression of Ang-1 was observed with immunocytochemistry and Western blotting methods.RESULTS: The result of sequencing showed that the cloned Ang-1 gene was consistent with the sequence reported in GenBank.After digestion with restrication endonuclease,the 1 512 bp fragment of Ang-1 gene and the 10.5 kb vector fragment of PNL-IRES2-EGFP were observed with gel electrophoresis.The insertion of Ang-1 gene in viral genome was confirmed.The EGFP expression was observed with the fluorescent microscope.In infected rMSCs,the mRNA and protein expressions of Ang-1 were confirmed.CONCLUSION: Lentiviral vector carrying Ang-1 gene has been successfully constructed.The infected rMSCs are able to express the Ang-1 mRNA and Ang-1 abundantly.This will facilitate the following exploratory development of Ang-1 gene-modified rMSCs.